Characterization of the cdhD and cdhE genes encoding subunits of the corrinoid iron-sulfur enzyme of the CO dehydrogenase complex from Methanosarcina thermophila
Characterization of the cdhD and cdhE genes encoding subunits of the corrinoid iron-sulfur enzyme of the CO dehydrogenase complex from Methanosarcina thermophila
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DOI：
10.1128/jb.178.2.340-346.1996
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发表时间：
1996-01-01
影响因子：
3.2
通讯作者：
Ferry, JG
中科院分区：
文献类型：
--
作者：
MaupinFurlow, J;Ferry, JG
嗜热Methanosarcina CO脱氢酶复合物含有一个由两个亚基（δ和γ）组成的类铁/铁硫酶。分别编码δ亚基和γ亚基的cddd和cdhE基因被克隆并测序。cdhD基因位于cdhE的上游，与cdhE相距3bp。这两个基因前面都有明显的核糖体结合位点。Northern （RNA）印迹和引物延伸分析表明，cddd和cdhE是由位于cddd上游数千个碱基的启动子共同转录的。推测的cddd和CdhE序列与从热醋酸梭菌铁硫酶β亚基和α亚基编码基因推断的序列相同37%。CdhE序列具有一个4 -半胱氨酸基序，有可能结合先前通过电子顺磁共振波谱在类铁/铁硫酶中发现的4Fe-4S粉尘。利用T7 RNA聚合酶/启动子体系在大肠杆菌中独立制备CdhD和CdhE，纯化后的CdhD蛋白与羟基钴胺素在碱基构型中重组。纯化后的CdhE蛋白显示出Fe-S中心和基离钴胺结合，其中苯并咪唑基氮原子不再是钴原子的下轴配体。
The CO dehydrogenase enzyme complex from Methanosarcina thermophila contains a corrinoid/iron-sulfur enzyme composed of two subunits (delta and gamma). The cdhD and cdhE genes, which encode the delta and gamma subunits, respectively, were cloned and sequenced. The cdhD gene is upstream of and separated by 3 bp from cdhE. Both genes are preceded by apparent ribosome-binding sites. Northern (RNA) blot and primer extension analyses indicated that cdhD and cdhE are cotranscribed from a promoter located several kilobases upstream of cdhD. The putative CdhD and CdhE sequences are 37% identical to the sequences deduced from the genes encoding the beta and alpha subunits of the corrinoid/iron-sulfur enzyme from Clostridium thermoaceticum. The CdhE sequence had a four-cysteine motif dth the potential to bind a 4Fe-4S duster previously identified in the corrinoid/iron-sulfur enzyme by electron paramagnetic resonance spectroscopy. A T7 RNA polymerase/promoter system was used to produce CdhD and CdhE independently in Escherichia coli, The purified CdhD protein was reconstituted with hydroxocobalamin in the base-on configuration. The purified CdhE protein exhibited an Fe-S center and base-off cobalamin binding in which the benzimidazole base nitrogen atom was no longer a lower axial ligand to the cobalt atom.